Hollow fiber membrane sample preparation devices

ABSTRACT

Simultaneous sample purification, enrichment and analysis of pharmaceuticals, illicit drugs, pollutants, biotechnological products, synthetic organic reaction products and food/flavor ingredients from complex matrices can be performed using porous hollow fiber or porous-disk liquid-membrane devices. The devices are part of a multi-well (e.g. 96-well) plate. The devices can be used for selective separation and enrichment of complex mixtures containing trace levels of analytes, and can be used in tandem with analytical instruments which routinely handle multiple samples under high throughput screening conditions. A multi-well/multi-vial plate can into state-of-the-art HPLC or GC sampling systems or LC/MS or GC/MS instruments. Samples can be enriched several orders of magnitude and can directly be withdrawn from the fiber and injected into the chromatographic instruments. Alternatively, these enriched samples can be introduced directly into MS, CE or other detection devices. Selective extraction of complex mixtures of analytes can be achieved through variation of acceptor phase chemistry, liquid membrane coating, pore size control of the hollow fibers, nature of the polymer from which the hollow fibers are made or pH of the acceptor phase.

RELATED APPLICATION DATA

This application claims the priority of U.S. Provisional PatentApplication No. 60/287,158, filed Apr. 26, 2001, herein incorporated byreference.

FIELD OF THE INVENTION

The invention in general relates to preparing samples for chemicalanalysis or synthesis, and in particular to systems, methods, andcompositions for performing simultaneous clean-up and enrichment ofanalytes of interest.

BACKGROUND OF THE INVENTION

Sample preparation, also termed pretreatment or clean up, is a pivotalstep in analytical method development for pharmaceuticals, illicitdrugs, food/flavor constituents, nutritional materials, environmentalpollutants and agricultural products such as pesticides, herbicides, andinsecticides. The scope of sample preparation is not restricted to theseareas of chemical analysis, and can extend to a wide range of otherfields of applicability such as synthetic chemistry, diagnostics andpurification of biotechnological products. In the arena ofpharmaceutical analysis, chromatography in general, and reversed phasehigh performance liquid chromatography (RP-HPLC) in particular, areextensively used for analyzing samples. Electrophoretic techniques havealso gained recognition as viable analytical tools. In this context,sample preparation ideally provides a reproducible and homogeneoussolution for injection into an analytical instrument such as achromatography column. Ideally, sample preparation also serves tofurnish a sample aliquot relatively free from interferences, preventscolumn damage, and is compatible with the intended analysis method. Theprecision and accuracy of the analysis method are frequently determinedby the sample preparation procedure.

SUMMARY OF THE INVENTION

The present invention provides methods, devices, and kits for performingclean-up and enrichment of analytes of interest. A sample purificationand enrichment method comprises: inserting a donor sample in a well of amulti-well plate, the donor sample comprising an analyte of interest;inserting a tubular hollow porous fiber into the well, the hollow fibercomprising a liquid extraction membrane, the hollow fiber enclosing aninternal cavity separated from the donor sample by the extractionmembrane; placing a static acceptor liquid in the internal cavity;simultaneously enriching and cleaning up the analyte of interest byextracting the analyte of interest from the donor sample, through theextraction membrane and into the acceptor liquid in the internal cavity,and transferring the analyte of interest and the acceptor liquid fromthe internal cavity to an analysis device.

A hollow-fiber membrane sample preparation multi-well plate forenriching and cleaning up samples comprises: a plurality of wells forholding a corresponding plurality of donor samples, each donor samplecomprising an analyte of interest; and a plurality of porous hollowfibers situated in the corresponding plurality of wells, each hollowfiber being situated in one of the wells, each hollow fiber including aliquid extraction membrane enclosing an internal cavity of the hollowfiber, for holding a static acceptor liquid within each hollow fiber toreceive the analyte of interest through the liquid extraction membraneinto the acceptor liquid.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing aspects and advantages of the present invention willbecome better understood upon reading the following detailed descriptionand upon reference to the drawings where:

FIG. 1-A shows an isometric view of a sample preparation multi-wellplate according to a presently preferred embodiment of the presentinvention.

FIG. 1-B shows a side sectional view of one of the wells and associatedpart of a top plate of the multi-well plate of FIG. 1-A.

FIG. 1-C is a side sectional diagram of a well and associated part of atop plate of an alternative multi-well plate, according to an embodimentof the present invention.

FIG. 1-D shows an isometric view of a top strip suitable for use with awell block such as the one shown in FIG. 1-A, according to an embodimentof the present invention.

FIGS. 1-E and 1-F show side sectional views of two wells and associatedparts of top plates according to other embodiments of the presentinvention.

FIGS. 2-A and 2-B show side sectional views of two wells and associatedparts of top plates according to other embodiments of the presentinvention.

FIG. 3 shows a side sectional view of a well and associated part of atop plate according to an embodiment of the present invention.

FIG. 4-A shows an isometric view of a sample preparation multi-wellplate comprising a plurality of tubes individually mounted on a supportblock, according to an embodiment of the present invention.

FIG. 4-B shows a side sectional view of one of the tubes of the assemblyof FIG. 4-A.

FIGS. 5-A through 5-C illustrate three different hollow fiber geometriesaccording to other embodiments of the present invention.

FIG. 6 is a schematic illustration of a vial holding a hollow fiber,according to an embodiment of the present invention.

FIG. 7 shows a side view of a device including a disk-shaped membranesupport according to an embodiment of the present invention.

FIGS. 8-A and 8-B show a side sectional view and a top view,respectively, of a vial cap or well cover including a collectioncontainer, according to an embodiment of the present invention.

FIG. 9 shows capillary electropherograms of a seven component basic drugmix after LPME demonstrating acceptor phase selectivity.

FIG. 10 shows capillary electropherograms of an eight component basicdrug mix after LPME to demonstrate acceptor pH selectivity.

FIG. 11 shows capillary electropherograms of a seven component basicdrug mix after LPME to demonstrate fiber selectivity.

FIGS. 12-A-B show chromatograms from the HPLC of a five componentacid/base drug mixture after LPME with four different membrane formingliquids to demonstrate membrane selectivity and selective extraction ofacidic and basic drugs with basic and acidic acceptors, respectively.

FIGS. 13-A-B show electropherograms of methamphetamine from human urineand plasma, respectively, after LPME extraction of the fluids containingthis drug.

FIG. 14 shows an electropherogram of naproxen after LPME extraction fromhuman urine.

FIG. 15 shows an electropherogram of citalopram and its metaboliteN-desmethylcitalopram from the plasma of a patient treated withcitalopram after LPME of the plasma.

FIG. 16 shows an electropoherogram of methamphetamine and citalopramfrom human whole blood after LPME.

FIG. 17 shows an electropherogram of tramadol enantiomers after LPMEfrom human plasma.

FIG. 18 shows an electropherogram of mianserine from the LPME of humanplasma.

FIG. 19 shows an electropherogram of five basic drugs from human plasmaand whole blood after LPME.

FIG. 20 shows an electropherogram of amphetamine from human urine afterLPME.

FIG. 21 shows an electroopherogram of chlorcyclizine from human plasmaafter LPME.

FIGS. 22-A-F show extraction profiles of promethazine, methadone andhaloperidol at different extraction times with 600 and 280 micron innerdiameter polypropylene fibers, for two sets of experiments.

DETAILED DESCRIPTION OF THE INVENTION

In the following description, it is understood that each recited elementor structure (e.g. plate) can be formed by or be part of a monolithicstructure, or be formed from multiple distinct structures. The statementthat a sample or liquid is static in a well is understood to mean thatthe sample does not flow through the well. A static sample may besubjected to agitation or vibration. The following descriptionillustrates embodiments of the invention by way of example and notnecessarily by way of limitation.

Sample matrices consist of products of organic, biological or inorganicorigin, and can exist in the form of solids, semisolids (includingcreams, gels, suspensions and colloids), liquids and gases. To cater tothe present generation requirements of trace level analysis and highthroughput screening, which involves several thousands of samples at atime, different device formats and sample preparation techniques havebeen developed, and are frequently automated. Such techniques includeliquid-liquid extraction, solid phase extraction, and supercriticalfluid extraction. Recent advances in these extraction methods includesolid phase microextraction, microwave-assisted solvent extraction,accelerated solvent extraction, derivatization protocols, liquid-liquidmicroextraction, and methods using molecularly imprinted polymers.

Liquid-liquid extraction (LLE) offers the benefits of quantitativerecovery, availability of a wide selection of solvents or combinationsof solvents, easier sample concentration after extraction, and highpurity that minimizes sample contamination. Liquid-liquid extraction maylead to emulsion formation, and may require time-consuming multipleextractions if the distribution constant between the organic and aqueousphases is low. Apart from miscibility considerations, an importantcriterion for solvent selection in liquid-liquid extraction is polarity.A variant of liquid-liquid extraction is micro-extraction, where anorganic solvent of density less than that of water is employed. Modernautosamplers are capable of performing such micro-extractionsautomatically on small volumes of aqueous samples in 2 mL vials.

Solid phase extraction (SPE) is currently one of the most popularmethods of sample pretreatment for pharmaceutical analysis. Unlike LLE,which is a one-stage separation process, SPE is a chromatographicprocedure resembling HPLC. SPE protocols normally consist of four steps:conditioning the packing, sample application, washing the packing toremove interferences, and recovery of the analytes of interest with moreconcentrated solvent. SPE devices encompass several formats, such ascatridge, disk and 48/96 deep well plates. Compared to LLE, SPE canallow more complete extraction of analytes, more efficient separation ofinterferences from analytes, reduced organic solvent consumption, easiercollection of total analyte fraction, more convenient manual operation,removal of particulates and easier automation. On the other hand, SPEmay be affected by variability of the bonded phases used in SPEcatridges, irreversible adsorption of some analytes, and leaching ofeither impurities present in the sorbents or of the bonded phasesthemselves. Irreversible adsorption of analytes can drastically lowerrecovery, while leaching can lead to contamination of the samplesolutions. With silica-based sorbents, an additional consideration isthe passage of fines through the frits used in the SPE catridges. SPEcatridges are normally meant for one time use only. Using SPE, samplesare normally preconcentrated by a factor of 2 to 4 only. For additionalenrichment, further evaporation of solvent is typically necessary.

Solid phase microextraction (SPME) is an offshoot of SPE. A typical SPMEmethod employs devices consisting of a fine, solid fused silica fibercoated with a polymeric stationary phase. The fiber is dipped into thesolution to be analyzed, and analytes diffuse to and partition into thecoating as a function of their distribution coefficients. Differentcoatings are commercially available for SPME-GC. Examples ofcommercially-available relatively less polar coatings includepolydimethylsiloxane (PDMS), and PDMS containing divinylbenzene(PDMS-DVB). Exemplary commercially-available, relatively more polarcoatings include polyacrylates (PA). Carbowax-DVB and carboxen-PDMSfibers have also been introduced recently. SPME is predominantlyemployed in environmental analysis in tandem with GC detectors. In SPME,the nature of the partition process is different from SPE or HPLC, andthe choice of fiber can be limited. Moreover, quantitation can bedifficult when several compounds are involved in competition in anunknown sample matrix.

The selective transfer of analytes or unwanted interferences across amembrane can be used to separate analytes of interest. Membranes used inseparation technology can be made from synthetic organic polymers,cellulose derivatives, or glass fibers, among others. Filtration andsolid-phase extraction disks represented the major areas of applicationsfor membranes in sample preparation until recently.

Analytes can be moved across membranes by diffusion as a result ofchemical or electrochemical gradients. Ultrafiltration, reverse osmosis,dialysis, microdialysis and electrodialysis are examples of techniquesthat utilize membranes for concentration, purification and separation ofanalytes. Membranes can be produced in many forms, such as sheet, roll,disk, capsule, cartridge, spiral-wound and hollow fibers. Semi-permeablemembranes allow passage of certain compounds, but not others, as in aflowing dialysis system.

Microporous semi-permeable membranes permit selective filtrationaccording to the size of their micropores. For example, molecular weightcutoff membranes allow passage of small molecules such as drugs, whileprecluding passage of large molecules such as proteins. Porouselectrically charged or ion-exchange membranes have pore walls withfixed positive or negative charges. The passage of ionic moleculesacross the membrane is governed by pore size and membrane charge. Indialysis with semi-permeable membranes, the sample (donor) solution isplaced on one side of the membrane and the acceptor solution is on theother side of the membrane. In some cases, interferences diffuse throughthe membrane, leaving a purified donor solution. More often, theanalyte(s) of interest pass through the membrane into the acceptorsolution, leaving interferences in the donor solution. For RP-HPLCanalysis, both the donor and acceptor liquids are usually water orbuffer.

Dialysis in a flowing system has also proved effective as an on-linesample preparation technique for the deproteination of biologicalsamples before HPLC analysis. The acceptor solvent is pumped to a traceenrichment column, which is later back-flushed into the HPLC instrument.These techniques have been automated and are in routine use inlaboratories.

Typical advantages of membrane-based systems developed to date overother sample preparation techniques include (a) reduced risk ofoverloading with sample or matrix components, (b) reduced contaminationand exposure to toxic or dangerous samples through the use of closedflow systems, (c) minimal use of organic solvents, and (d) easyautomation in flow systems. At the same time, membranes can be subjectto fouling by particulates or macromolecules. Such fouling can result inflow rate decreases and diminished membrane effectiveness.

Sample Preparation Using Supported Liquid Membranes (SLM):

Supported-liquid membrane (SLM) enrichment techniques can be thought ofas combining aspects of dialysis and liquid-liquid extraction. In oneimplementation, a porous membrane support is impregnated with awater-insoluble organic solvent and is placed in a mounting block.Compounds are extracted from the donor side into the membrane as afunction of their solubility in the supported liquid, where they arethen re-extracted from the membrane into the acceptor side. A simpleexample of the use of this technique is the enrichment of a carboxylicacid from an aqueous donor solution. By adjusting the pH of the donorsolution below the pK_(a) value of the acid, the ionization of thecarboxylic acid is suppressed, allowing the nonionic form to beextracted into the immobilized liquid on the membrane. The non-ionizedacid diffuses through the membrane to the acceptor side, which has abasic pH where the organic acid is extracted in its ionized form.Therefore, the carboxylate anion is concentrated since it no longer canreextract into the membrane. Enrichment factors of several hundred canbe achieved using a support liquid membrane sample preparation method ofthe present invention, as described in the Examples below. Placing asorbent trap or precolumn between the membrane device and the HPLCinstrument allows the analyte to be concentrated ever further.

Sample separation and enrichment using supported liquid membranes onporous hollow fiber supports employ a porous hollow fiber or combinationof several fibers, a liquid membrane supported in the pores of thefiber, a sample enriching acceptor solvent/solution, and a device formatfor introducing the sample and acceptor solutions into different regionsof the fiber. The device format enables partitioning and enrichment ofthe analyte under investigation, and transfer of the analyte-enrichedacceptor solution into an analytical instrument for quantitation.

Microporous supports useful for incorporating membrane forming solid orliquid materials are known in the art. A hydrophobic microporous supportis a material that is not spontaneously wetted by water and has anopen-celled, inter-connected structure. Such a microporous supportshould be composed of materials that are compatible with the solid orliquid membrane substance used for coating. Examples of materialssuitable for such supports include polyolefins, polysulfones,polytetrafluoroethylene, polycarbonates, polyetherketones, polystyrene,cellulose, cellulose acetate and other polymeric materials. The pores ofcommercially available microporous materials are in the range of about0.02 to about 2 microns in effective diameter. Pores as small as 0.01micron and as large as 10 microns are not unusual and a specific poresize is not necessarily important in a given application. Typically,commercial porous support thickness values range between 10 and 300microns, although thicker supports are used for certain applications.The porosity of supports is ideally sufficient to provide an opennetwork through the support. Typical commercial fibers have a porosityof about 30 to 80%. A commercially-available Celgard® polypropylenemembrane, for example, has a porosity between 40 and 50%. Porosity isdefined as the fractional volume of the membrane that is open ratherthan substrate material. Supports may be treated to alter their surfaceproperties. For example, polyethylene films may be treated with chromicacid to render the films less hydrophobic. Hollow fiber format ofsupports (in comparison with a flat sheet), especially in helical orspirally wound formats, provide a high ratio of support surface area tovolume of the sample solution and acceptor solution.

For aqueous sample solutions, the supported liquid membrane is typicallya water immiscible organic solvent. When a sample solution consists ofanalytes dissolved in organic solvent, the membrane is typically anaqueous-based system. Since sample pretreatment predominantly involvesaqueous solutions, the supported membranes are typically chosen fromaliphatic or aromatic hydrocarbons, ethers, nitrites, aldehydes orketones, and alcohols which are immiscible with water. Some specificsuitable membrane liquids include 1-octanol, 2-octanone, diphenyl ether,nitrophenylalkylethers ranging from pentyl to decyl for the alkyl part,higher alkylpyridines such as 4-(1-butylpentyl) pyridine,1-octyl-2-pyrrolidone, benzonitrile, diisopropylbenzene, cyclohexanone,tri-n-butylphosphate, triglycerides with alkyl chain lengths of 6 to 24carbon atoms and fatty acid esters of cholesterol with alkyl chainlengths of 2 to 20 carbon atoms, to mention a few examples. Membranestability tends to improve when an extremely hydrophobic liquid such asdodecane is used, but very little flux is produced owing to lowdiffusion coefficients in such liquids. On the other hand, polarsolvents tend to afford high diffusion coefficients, but have lowstability. To balance these factors, it is desirable to use a mixture ofsolvents. Most membranes have lifetimes of five days or less. Withnitrophenyloctyl ether, membrane lifetimes of 10-20 days have beenobserved. Suitable surfactants may also be used to enhance the stabilityof mixed solvent membranes, as for example, nonionic surfactants withhydrophilic-lipophilic balance ranging from 8 to 15, such aspolyoxyalkylene esters or ethers.

Polymeric membranes formed either by polymerization of monomers in thepores of support materials, or by coating preformed polymers dissolvedin appropriate solvents, have been found to be significantly more stableand also exhibit high partition coefficients towards small organicmolecules. Examples of such polymers include polyalkylene glycols,polyvinylpyrrolidones, polyesters, polyurethanes and functionalizedpolyolefins. Polydimethylsiloxane membranes were reported to demonstrateselectivity for higher alcohols compared to ethanol.

The acceptor liquid contacts the outer shell surface of a hollow fibermembrane when the sample solution is passed through the lumen side ofthe fiber. Conversely, if the sample solution is circulated through theshell side, the acceptor solution is passed through the lumen side.Acceptor solutions can be aqueous, basic, or acidic solutions, orpolymers in the liquid state such as polyethylene glycol, depending onthe type of application. Acceptor solutions can also include complexingagents capable of forming a complex with the analyte(s) of interest.

Several flow-through systems employing liquid membranes supported onhollow fiber supports have been described. Commonly, provision is madefor sample (feed) flow from the shell side of the fibers, as well as foracceptor solution (strip solution) flow across the lumen side of thefiber, both involving pumping systems. For information on known hollowfiber systems see for example U.S. Pat. Nos. 4,666,543, 5,282,964,5,474,902, 5,846,427, 5,202,023, and 5,252,220.

According to the preferred embodiment of the present invention, samplepreparation systems and methods can employ a variety of sampling deviceformats incorporating supported liquid membranes contained in the poresof polymeric hollow fibers. Such devices are capable of simultaneouslyeffecting clean up and enrichment from trace/impure state of a sample toseveral orders of magnitude more concentrated and purified condition. Inparticular, such devices are capable of providing microliter levelvolumes of pure extracts, which are not commonly obtainable directly bystandard sample preparation techniques. Furthermore, these devices areamenable for integration into state-of-the-art automated chromatographicand mass spectrometric instrumentation used for high throughputscreening of pharmaceuticals and other types of analytes.High-throughput screening involves the automated analysis of largenumbers of samples within short time frames. Samples can be purified andenriched directly on the autosampler systems of these analyticalinstruments, and aliquots from these enriched and purified samplesolutions can be injected directly into the chromatographic columns ormass spectrometer.

According to the preferred embodiment of the present invention, thesampling device formats comprise 48 or 96 or 384 well plate blockscarrying hollow fibers suspended in each well. Extraction/purificationcan be carried out by automatic or manual delivery of sample solutionsinto the shell side of the fibers in each well. The fibers carry theacceptor (or strip or extracting) solution on the lumen side andanalytes diffuse through the supported liquid membrane in the pores ofthe fibers into the acceptor solution. The autosampler injector needleof the chromatographic instrument can pick up the enriched sampledirectly from the fiber and deliver it to the instrument for analysis.The well plate assembly can be mounted directly onto the analyticalinstrument.

Alternatively, the sample enrichment process can be carried out inautosampler vials, which are commonly used in liquid chromatographicinstruments. A miniature device can incorporate hollow fibers into eachof the vials individually. The ends of these fibers can be connected toappropriate inlet/outlet ports located in the cap portion of the vialsfor automated delivery and withdrawal of acceptor solution before andafter enrichment, respectively. Thus, the devices provide for enrichingand analyzing multiple samples at a time through an automated samplingsystem.

The fibers suspended in the well plates may be modified by severalpermutations and combinations of parameters to incorporate selectivityfeatures which would permit the isolation of a single analyte from acomplex mixture or a group of analytes from other groups or excludeunwanted materials from human fluids or synthetic reaction mixtures.Thus, fibers made from different polymeric materials (such aspolypropylene, polysulfone, polycarbonate or polyether sulfone, etc.)can be suspended in the wells to harness selectivity arising from fiberchemistry. Alternatively, the fibers can be coated with differentmembrane forming liquids to utilize membrane-based selectivity foroptimization of enrichment and selective extraction. The chemistry ofthe acceptor solutions (strong or weak acids or bases, for example) aswell as the pH of the acceptor solutions can be varied along withvariation of the fiber chemistry, to achieve the desired separation.Furthermore, the pore size of the fibers can also be varied to effectselective diffusion into the fiber.

With a multiwell format, all these variables can be incorporated intoone and the same well plate block. This feature enables the probing ofseveral selectivity-imparting parameters simultaneously to arrive atoptimal conditions for a desired separation/purification. Making use ofa host of acidic, basic and neutral pharmaceuticals in wide circulationaround the world, the performance of the device is demonstrated withrespect to enrichment, selective extraction and speed of analysis ofmultiple samples.

The devices of the present invention operate in the static mode. Samplesolution to acceptor liquid ratios ranging from 20 to 200 can beemployed. Thus, one has the choice of using sample solution volumes aslow as 500 μL to as much as 10 mL. Furthermore, the extractions can becompleted in about 15 minutes and sample enrichments ranging from 30 to200 fold can be achieved. Such high levels of sample enrichment are notcommonly achievable with presently-available standard sample preparationtechniques.

Systems and methods according to the preferred embodiment of the presentinvention address shortcomings of state-of-the-art sample preparationtechniques such as liquid-liquid extraction, solid phase extraction andsolid phase micro-extraction. These techniques may not easily handlevery small volumes of solvents, e.g. less than 100 microliters ofsolvent used as extracting medium. Analysts may prefer to use such smallvolumes during sample preparation in order to achieve a high degree ofsample enrichment, while effecting complete extraction simultaneously.Typically, these techniques use larger volumes of sample. A subsequentconcentration and/or reconstitution step is then used to generatedetectable sample levels for analysis. In a number of instances,extraction is incomplete when these techniques are used, which leads toproblems in quantitation.

Membrane-based separations or extractions are particularly attractivesince these approaches can use small volumes of solvents and canwithstand extremes of pH, unlike silica-based solid phase extractionsystems. Polymeric materials used in solid phase extraction, such asOasis®, can overcome this pH problem, but may not exhibit a widespectrum of selectivity and are not universal with respect to solventcompatibility. In the prior art, both silica and polymer based solidphase extraction systems are available in the well plate format and havebeen used for sample purification during high throughput screening ofpharmaceuticals. However, problems such as contamination due to leachingfrom the sorbents and strong retention of analytes (sometimesirreversible retention) can persist.

According to the preferred embodiment of the present invention, hollowfiber membranes can be used as devices for obtaining sample enrichmentof several orders of magnitude, both with small organic molecules andwith complex biomolecules such as proteins and nucleic acids. Fibers ofdifferent chemistries or different acceptor solutions or differentliquid membranes can be employed in one and the same device format. Awide spectrum of selectivity can be achieved by employing differentfiber/membrane chemistries and variation of acceptor phases or pHsimultaneously.

Simple supported liquid membrane hollow fiber devices can be employed inthe well plate and autosampler vial formats in a static mode that canfurnish a high degree of sample enrichment. The devices can be operatedinterchangeably either in the manual or automated modes and providerapid screening of large volumes of samples. These devices can functionas either two-phase extraction systems (aqueous feed solution on theshell side of the fiber and organic solvent acceptor solution on thelumen side, with the same solvent forming the supported membrane) or asthree phase extraction systems (aqueous feed, supported liquid membranephase and an aqueous acceptor phase) during the sample enrichmentprocess.

Theory:

The theoretical discussion below is intended to generally illustrateparticular embodiments of the present invention, and is not intended tolimit the scope of the invention to the described illustration.

Consider a hollow fiber membrane, two-phase liquid phasemicro-extraction (LPME) system having donor and acceptor phases, withthe supported liquid membrane being the same material as the acceptor.When an analyte attains concentration equilibrium between the two phases(see equation 1), extraction is complete. For an analyte A, thedistribution between the two phases is governed by Nernst's distributionlaw, as given in equation 2.A(donor)⇄A(acceptor)  (1)K _(a/d) =C _(eq[a]) /C _(eq[d])  (2)

In eq. [2], C_(eq[a]) is the equilibrium concentration of A in theacceptor phase and C_(eq[d]) is the concentration of A in the donorphase. By the law of conservation of mass, the initial mass of theanalyte (n_(i)) is equal to the sum of the individual quantities of theanalyte present in the two phases, as in equation 3.n _(i) =n _(d) +n _(a)  (3)

At equilibrium, eq. 3 can also be written asCiV _(d) =C _(eq[d]) V _(d) +C _(eq[a]) V _(a)  (4)where C_(i) is the initial concentration and V_(d) and V_(a) are thesample phase volume and acceptor phase volume, respectively. The amountof analyte extracted into the acceptor phase of the system can becalculated by substituting K_(a/d)C_(eq[d]) for C_(eq[a]) in equation 3.n _(eq) =K _(a/d) V _(a) C _(i) V _(d)/(K _(a/d) V _(a) +V _(d))  (5)

Then, the recovery (R) can be expressed by equation 6 below, while theenrichment (E) can then be calculated by equation 7.R=n _(eq)×100/C _(i) V _(d)=(K _(a/d) V _(a)×100)/(K _(a/d) V _(a) +V_(d))  (6)E=C _(a) /C _(i) =V _(d) R/V _(a)×100  (7)

Equation 7 can be used to calculate the theoretical recoveries andenrichment for both LLE and LPME. For the three phase system (donor,supported liquid membrane, acceptor), the mass balance can be expressedby eq. 8:n _(i) =n _(d) +n _(org) +n _(a)  (8)where n_(org) represents the mass of analyte in the supported liquidmembrane. Equation 8 can also be expressed asCiV _(d) =C _(eq[d]) V _(d) +C _(eq[org]) V _(org) +C _(eq[a]) V_(a)  (9)where C_(eq[org]) corresponds to the concentration of analyte in thesupported liquid membrane at equilibrium and V_(org) is the volume ofthe organic membrane phase.

In a three phase system, there will be two distribution constants whichrepresent the two equilibria occurring in this system, as given inequations 10 and 11, respectively.K _(org/d) =C _(eq[org]) /C _(eq[d])  (10)K _(a/org) =C _(eq[a]) /C _(eq[org])  (11)

K_(a/d) can be computed by equation 12:K _(a/d) =K _(org/d) +K _(a/org)  (12)from which we can derive equation 13:K _(a/d)=(C _(eq[org]) /C _(eq[d]))(C _(eq[a]) C _(eq[org]))=C _(eq[a])/C _(eq[d])  (13)where K_(org/d), K_(a/org) and K_(a/d) are the distribution constantsbetween the pair of phases organic and donor, acceptor and organic andacceptor and donor, respectively.

The amount of analyte extracted into the acceptor phase of the systemcan be calculated by substituting K_(a/d)C_(eq[d]) for C_(eq[a]) inequation 9. Rearrangement leads to equation 14:n _(eq)=(K _(a/d) V _(a) C _(i) V _(d))/(K _(a/d) V _(a) +K _(org/d) V_(org) +V _(d).  (14)Then, the recovery R can be expressed asR=(n×100)/(CiV _(s))=(K _(a/d) V _(a)×100)/(K _(a/d) V _(a) +K _(org/d)V _(org) +V _(d)).  (15)Finally, the enrichment E can be calculated using equation 16:E=C _(a) /Ci=(V _(d) ×R)/(V _(a)×100).  (16)

ince the three phase system in LPME involves back extraction, thistechnique differs from LLE in that the extraction of the sample from thematrix into the organic phase and then from the organic phase into theacceptor phase occurs simultaneously in LPME, while it is a two stepprocess in LLE. However, equations 15 and 16 can be used to predictrecovery and enrichment in both processes approximately.

Preferred Device Formats/Geometries:

FIG. 1-A shows an isometric view of a multi-well plate 20 according to apresently preferred embodiment of the present invention. Plate 20comprises a 96-well block 22, and a top plate 24 secured to block 22.Block 22 comprises an array of evenly-spaced sample wells each having atop opening. Top plate 24 comprises a plurality of analyte-collectionthrough holes (apertures) 26, each corresponding to one of the wells ofblock 22.

FIG. 1-B shows a side sectional (1-1′) view of an exemplary well 30 ofblock 22, and the corresponding part of top plate 24 extending over well30. The top of through hole 26 includes a cone-shaped tapered surface34, and functions as an inlet/outlet port. The tapered shape of surface34 facilitates the entry of a transfer device such as a needle into hole26. Top plate 24 comprises a projected tubular end 32 extending downwardinto well 30. Projecting end 32 can be integrally formed as one piecetogether with the planar part of top plate 24, or can be a separate partsuch as a resin tube attached to the planar part of top plate 24.

A hollow fiber 36 in a single-rod format is connected to projected end32 along its top open side. The connection between the projected end 32and the fiber 36 can be effected by well-known technologies, such asbinding with an adhesive or by direct extrusion of fiber 36 through end32. A bottom end 40 of fiber 36 is closed. Bottom end 40 is preferablyformed by a sealant such as an adhesive or a plastic. To seal bottom end40, an open-ended fiber may be placed on a heated plastic surface.Liquid plastic then enters the fiber through the open end, and cools toclose the fiber end. An interior cavity 38 formed within hollow fiber 36is separated from any liquid present in well 30 by the wall of fiber 30.

The projected end 32 with the connected fiber 36 fits into thecorresponding well 30 of block 22. The entire top plate 24 and the wellblock 22 can be locked or secured to each other by a hook mechanism. Thecontact surface between top plate 24 and well block 22 is preferablytight fitting, so as to create a sealed environment. For ease ofmanufacturing and quality control each top through hole 26 ispreferentially located coaxially with the center of the correspondingwell 30. Well 30 can be designed to hold different volumes by varyingits depth, such that different lengths of fiber 36 are accommodatedwithin well 30.

A sample solution is dispensed into well 30 with an auto-dispenser ormanually, as desired. An acceptor liquid or solution is then injectedinto cavity 38, on the lumen side of hollow fiber 36 with the injectorsystem of a liquid chromatograph or any other robotic system. Prior toinjection of the acceptor solution, fiber 36 is preferably precoatedwith the membrane-forming solution. After a specified period of time,the enriched and purified analyte solution can be sampled directly fromcavity 38 through the same outlet 26, by the same auto-sampler orrobotic system. A vibrator can be located underneath the well block 22to assist in the efficient extraction of the analyte(s) of interest fromthe sample into the acceptor solution.

In an alternative embodiment depicted in FIG. 1-C, provision is made foran additional conically shaped sample inlet through-hole 46 definedthrough a top plate 24′. Through hole 46 extends over well 30 along anarea external to fiber 36, such that hole 46 is not in directcommunication with cavity 38. Hole 46 serves as an inlet forauto-dispensing of the sample solution to well 30 while plate 24′ ismounted on well block 22. Hole 46 can also be useful when viscoussamples, not injectable by autodispenser needles, or suspension-typesamples are to be introduced into the well, as for example aqueous-oilmixtures. Such viscous samples can be inserted through hole 46 using apipetter or other suitable device. Alternately, if a microscale organicsynthetic reaction is carried out in the well, hole 46 can be used tointroduce reagents into well 30. The reaction product can diffusethrough membrane 36 into cavity 38, and can be analyzed directly. Sucharrangements are especially useful in separating tritylated productsfrom unreacted oligonucleotides.

As shown in FIG. 1-D, one or more strip-shaped partial top plate(s) 48can be used instead of a global (x-y) top plate such as the top plate 24shown in FIG. 1-A. Using partial top plate 48 can be convenient if, forexample, only one segment of eight or twelve well plates, but not theentire cross section of ninety six wells, is needed for an extraction.Partial top plate 48 can carry twelve holes 26 evenly distributed in thesame row with one single hollow fiber 36 connected to each of the holes26 in this row. The fiber suspension arrangement for each hole 26 issimilar to the one described with reference to FIG. 1-A and 1-B. A sealmat (not shown) can be used to cover the open well holes of block 22 notcovered by top plate(s) 48.

FIG. 1-E shows a side sectional view of another geometry for a samplepreparation plate 120 according to the present invention. Plate 120comprises a 96-well block 122 fitted with a top flexible seal mat 124.Seal mat 124 can extend over the entire top surface of block 122. Sealmat 124 can also form an elongated strip extending over a single row ofwells formed in block 122. Block 122 includes 96 evenly distributedwells 130, one of which is shown in FIG. 1-E. A seal stud 134individually extends and tightly fits into each respective top opening135 of a well 130. Seal stud 134 forms part of seal mat 124. An open endof a precoated single hollow fiber 36 is connected to a tube 137 whichpasses through seal stud 134. A support material 141 such as a resin maybe filled around tube 137 so as to position tube 137 rigidly within well130. Support material 141 may be the same material used for the flatpart of seal mat 124 and/or seal stud 134. The top end of tube 137 isconnected to a funnel 139. Funnel 139 facilitates the access of atransfer device such as a needle to the interior cavity defined withinfiber 36. Tube 137 and funnel 139 can be formed of a material such as aplastic or a metal such as stainless steel.

As illustrated in FIG. 1-F, an injection port 46′ can be provided alongthe bottom of a well 30 defined in a multi-well block 23. To insert asample into well 30, the plate is flipped over such that injection port46′ is positioned along the top surface of the plate. A sample isinjected through port 46′ manually or automatically, port 46′ is sealed,and the plate is flipped back to the position illustrated in FIG. 1-F.The acceptor solution is then injected through hole 26, and after adesired period of time the analytes of interest are extracted throughhole 26. The geometry of FIG. 1-F facilitates forming block 22 and upperplate 24 as a single monolithic piece.

As shown in FIG. 2-A, a tubular, tapered fiber-protecting insert 80 canbe disposed laterally around fiber 36, in order to protect fiber 36 fromcontact with external structures during the assembly or operation of thesample preparation plate. Such contact can result in the crumpling,twisting, or collapse of fiber 36. Insert 80 can form part of upperplate 24. Insert 80 can be integrally formed as one piece together withthe planar part of upper plate 24, or can be attached to the planar partof upper plate 24 using for example an adhesive, a fastener, or a pressfit. Insert 80 hangs down from the lower flat surface of upper plate 24into well 30, and is centered around fiber 36 and aperture 26. Insert 80is longer than the longitudinal extent of fiber 36, such that insert 80extends below fiber 36. The bottom end of insert 80 is open, so as toallow the liquid within well 30 to contact fiber 36. Insert 80preferably has a downward-narrowing tapered shape. The tapered shapefacilitates the entry of insert 80 into well 30. Insert 80 preferablyhas an annular upper contact section 82 situated along the flat surfaceof upper plate 24. Contact section 82 is sized to fit snugly within well30, such that a press fit is established between insert 80 and wellblock 22 along the surface of contact section 82 when block 22 and upperplate 24 are fully engaged together.

In addition to insert 80, a global protective sidewall can be providedalong the external boundary of upper plate 24, in order to provideglobal mechanical protection to all fibers 36. Insert 80 can alsoinclude round perforations extending through the surface of insert 80,so as to allow the fluid flow across insert 80. The perforationsize/diameter can be on the order of millimeters or less, such thatfluid can flow unimpeded through insert 80, while fiber 36 remainsmechanically protected from outside contact. As illustrated in FIG. 2-B,insert 80 can be provided as part of an intermediate plate 90 stackedbetween a top plate 24′ and well block 22. Intermediate plate 90 canalso be thought of as forming part of the top plate.

As illustrated in FIG. 3, one or more elongated alignment/protectionpins 92 a-b can be provided as part of an upper plate 24″, for each well30 of a well block 22″. Pins 92 a-b preferably extend along at least theentire length of fiber 36, and are disposed around fiber 36. Pins 92 a-bensure the alignment of each fiber 36 within its corresponding well 30,and laterally protect fiber 36 from contact with external structures.Each pin 92 a-b fully fits through a corresponding longitudinal guidinghole 94 a-b defined within block 22″. Guiding holes 94 a-b are locatedbetween adjacent wells 30. An annular seal flange 96 can be providedalong the top of fiber 36, for sealing well 30 when upper plate 24″ isfully engaged to well block 22″. Seal flange 96 forms part of upperplate 24″. Seal flange 96 is attached to the bottom surface of theplanar part of upper plate 24″, and is centered around fiber 36. Theexternal lateral surface of seal flange 96 is sized to fit snugly withinwell 30.

FIG. 4-A shows an isometric view of a multi-well plate 220 according toan alternative embodiment of the present invention. Plate 220 comprisesa receiving base plate 222 having an array of holes definedtherethrough, and a plurality of tubes (cartridges) 252 mounted on baseplate 222. Each tube 252 is mounted through one of holes defined in baseplate 222. Preferably, the cross section of each tube 252 is circular orsquare-shaped.

FIG. 4-B shows a side sectional view through an exemplary tube 252 and apart of base plate 222 below the shown cartridge 252. Base plate 222 mayinclude a plurality of individual wells each corresponding to each tube252, or a global cavity situated underneath the holes of plate 222,common to all tubes 252. Each tube 252 can be connected to base plate222 pressing or screwing type of fit or any other connection mechanism.A bottom base block 258 may be used to support base plate 222, and toprovide a desired height to plate 220, such that plate 220 optimallyfits into an auto-sampler assembly of a liquid chromatographic system.

Each tube 252 includes a tube body 254 for holding a sample of interest,and a cap 256 mounted on top of tube body 254. An internal collectioncavity 38 is defined within a hollow fiber 36, as described above withreference to FIGS. 1-A and 1-B. Hollow fiber 36 preferably hangs from adownward-protruding tubular stud structure 232 of cap 256. Cap 256includes a top central opening 226 for providing access to cavity 38.Top cap 256 can be replaced with a partial plate (not shown) withmulti-holes having appropriately connected fibers in the bottom. Thepartial plate can be inserted to cover a segment defined by multipletubes 252. A seal strip and seal cap can be applied to any open tubes252 not covered by this partial plate

The bottom end of fiber 36 can be closed, as shown in FIG. 4-B. Thebottom end of fiber 36 can also be mounted on a second stud (not shown)positioned underneath stud 232, such that fiber 36 is held between thetwo studs. Fiber 36 then has two open ends: an upper inlet and a loweroutlet. In such an arrangement, the acceptor solution containing theanalyte(s) of interest can be collected through a bottom aperture of thetube, into a corresponding well positioned underneath the bottomopening. The bottom aperture of the tube is connected to the second(lower stud). The bottom aperture is held closed while the analyte ofinterest collects inside the fiber volume, and is opened after a periodof time in order to allow the acceptor solution to flow out.

As in the monolithic multi-well plate described with reference to FIGS.1-A and 1-B, a sample solution is disposed in each tube 252 with anauto-dispenser device. An acceptor solution is injected through theinlet/outlet hole 226 into the cavity 38 defined within hollow fiber 36,using an auto-sampler injector or robotic system. The acceptor solutioncontaining the extracted sample in cavity 38 can be drawn off throughthe same inlet/outlet hole 226 by the auto-sampler or robotic system.

FIG. 5-A illustrates part of a sample preparation plate 320 according toanother embodiment of the present invention. Plate 320 includes amulti-well block 322, and a top plate 324 mounted on and covering block322. An inlet adaptor 343 is built on the top plate 324, above a well330. Inlet adaptor 343 has an open top inlet 349, and two bottom outlets344, 345 arranged orthogonal to each other. A single U-shaped hollowfiber 336 has two open ends 347, 348 respectively connected to outlets345, 344.

The illustrated hollow fiber geometry facilitates a reduction in theformation of air bubbles inside hollow fiber 336. As acceptor liquid isinserted vertically into well 330 through the fiber segment aligned withinlet 349, the air initially present within fiber 336 can escape upwardthrough the other segment of fiber 336, away from the incoming liquid.The shown geometry also facilitates uniform distribution of the acceptorliquid within hollow fiber 336. The illustrated U-shaped hollow fibergeometry also allows the use of a hollow fiber 336 longer than the depthof the corresponding well 330.

FIG. 5-B illustrates part of a sample preparation plate 420 according toanother embodiment of the present invention. Plate 420 includes amulti-well block 422, and a top plate 424 mounted on block 422. AnH-shaped adaptor 443 is formed through top plate 424, above a well 430.Adaptor 443 includes two inlet ports 449, 449′ and two correspondingoutlet ports 445, 445′. Outlet ports 445, 445′ are respectivelyconnected to two rod-shaped hollow fibers 436, 436′. The illustratedhollow fiber geometry facilitates different kinds of possible extractioncombinations such as forward and back extractions. The horizontal tubeof adaptor 443 serves as an air vent.

FIG. 5-C illustrates part of a sample preparation plate 520 according toanother embodiment of the present invention. Plate 520 includes amulti-well block 522, and a top plate 524 mounted on block 524. Twoparallel tubes 557, 558 project vertically through top plate 524, abovea well 530. A U-shaped hollow fiber 536 has two open ends 555, 556connected to tubes 557, 558, respectively. The top ends of the tubes557, 558 to a small funnel 559 and an open cap 560, respectively, whichare positioned on the topside of the plate 524. The funnel 559 can serveas an inlet and/or outlet port. The open cap 560 can function as anoutlet, a pressure equalizer, or a vent for any air inside hollow fiber536. As described above, tubes 557, 558 can be formed from a plastic ora metal such as stainless steel, among others.

FIG. 6 is a schematic illustration of part of a sample preparation plate620 according to another embodiment of the present invention. Plate 620comprises individual vials 652 mounted in corresponding wells 630defined in a base plate 622. Each well 630 can carry vials of differentsizes and or volumes—for example, 4 mL or 2 mL volumes. Each vial 652comprises a tubular vial container 654 defining a well 630 for holding asample of interest, a vial cap 656 mounted on vial container 654, and ahollow fiber sample preparation structure 636 hanging from vial cap 656into well 630. Vial cap 656 can be press fitted, screw fitter, orotherwise fastened by known means to vial container 654. Vial cap 656and the attached hollow fiber structure 636 can have any of thegeometries illustrated in FIGS. 1-B through 5-C. In the arrangementshown in FIG. 6, the collection of individual vials 652 effectivelyforms a modular sample preparation multi-well plate, while thecollection of vial caps 656 of vials 652 effectively forms a modularfiber membrane support supporting a plurality of hollow fiber membranesdisposed in the wells of the multi-well plate.

FIG. 7 shows part of a multi-well plate 720 according to anotherembodiment of the present invention. Plate 720 comprises a multi-wellblock 722 defining a plurality of collection wells 729, and amulti-aperture top plate 724 mounted on block 722. A pre-coateddisk-shaped porous membrane support 771 is mounted in a deep counterhole774 defined within top plate 724. Membrane support disk 771 rests on anannular protrusion that prevents disk 771 from sliding downward. Asample-holding well 730 is defined in the area enclosed by top plate 724and situated above disk 771. To perform LPME in the well shown in FIG.7, the sample of interest is preferably placed in sample-holding well730, while the acceptor solvent is placed in collection well 729 so asto contact disk 771. After the analytes of interest have passed fromsample holding well 730 into collection well 729 through the liquidmembrane defined in the pores of disk 771, top plate 724 is removed andthe enriched and purified analytes of interest are collected fromcollection well 729.

FIGS. 8-A and 8-B shows side sectional and top views, respectively, ofpart of a sample preparation plate 820 according to another embodimentof the present invention. An annular support structure 824 is mounted ina centered position above a sample-bolding well of a multi-well block(not shown). Support structure 824 can be a vial cap as illustrated inFIGS. 6 and 4-A-B, or form part of a partial or whole top cover plate asillustrated in FIGS. 1-A through 5-C.

A concave, U-shaped or V-shaped collection microcontainer 876 isdisposed below a collection aperture 826 defined in support structure824. Microcontainer 876 can preferably hold up to 100 microliters of afluid. One end of a U-shaped hollow fiber 836 is connected to an inlettube which passes through an opening 877 provided in support structure824 into the sample-holding well. The other end of hollow fiber 836 isconnected to an inlet of a collection tube 878. An outlet of collectiontube 878 is disposed in/above collection microcontainer 876. Hollowfiber 836 is held by its end connections to the inlet and collectiontubes, and hangs into the sample-holding well so as to contact a sampleof interest held in the sample-holding well.

Acceptor fluid is inserted into hollow fiber 836 through inlet tube 877.After the analytes of interest have passed from the sample-holding wellinto the acceptor fluid held in hollow fiber 836, positive pressure isapplied through inlet tube 877 so as to evacuate the contents of hollowfiber 836 through collection tube 878 into collection microcontainer876. The enriched and purified analytes of interest can then be removedfrom collection microcontainer 876 using an autosampler needle of achromatographic instrument or other known devices.

The plate geometry illustrated in FIGS. 8-A and 8-B reduces the chancethat an autosampler needle used for collecting the acceptor solutiondamages hollow fiber 836. The illustrated geometry allows increasedflexibility in the choice of hollow fiber membrane shapes and internaldiameters, and in the diameter of the needle used to collect theanalytes of interest.

The single vials described above can be used individually or mounted ona sampler plate on a LC instrument. Such single vials can be produced byfitting commercially available or custom made products with thedescribed hollow fiber membranes and associated support structures.Alternative formats and/or modifications can be visualized by thoseskilled in the art relating to the above detailed descriptions. Suchmodifications should be deemed to be within the same scope of thepresent invention.

Separation and Enrichment of Pharmaceuticals:

The description below focuses on the application of the multiplesampling devices described above to the extraction of trace levels ofpharmaceuticals and other small molecules in aqueous media or biologicalmatrices using 10 to 50 microliter volumes of acceptor (or strip) phase,preferably 25 microliters, to obtain optimal enrichment. Such enrichmentis useful in producing measurable signals by the analytical instrumentsutilized for the analysis of pharmaceuticals at the nanogram or picogramlevel, especially when dealing with mixtures of analytes. Suchanalytical instruments can include high performance liquidchromatographs, gas chromatographs, capillary electrophoreticinstruments, mass spectrometric detectors, and others. Sample volumes of500 μL or even less from the lower end up to 25 mL on the higher end canbe utilized for extraction and enrichment with the well plate or vialforms of devices disclosed in the current invention. When the analytesunder investigation are basic drugs, the sample solutions are treatedwith a base such as sodium hydroxide or ammonia to bring the pH of thematrix to around 7.0 or over. These basic analytes will then exist asfree bases and not in the form of salts, to facilitate extraction acrossthe membrane barrier. Conversely, if the matrix contains acidicpharmaceuticals, its pH is adjusted to be around 2.0 to 5.0 so that theacids exist in the free state and do not form carboxylate anionstructures. For the extraction and enrichment of basic analytes, acidicacceptor solutions are used, as for example, 0.1M hydrochloric acid oracetic acid. For acidic analytes, 0.1M sodium hydroxide or sodiumcarbonate could be utilized.

The supported liquid membrane layer could be predeposited on to thehollow fiber prior to fitting of the fiber into the device, for coatingsthat can form stable membranes. Polymeric membranes are quite stableover long periods of time and have excellent diffusion characteristicsfor a wide range of analytes. By the same token, monomeric materials canbe initially introduced into the pores of the hollow fiber and thenpolymerized in situ. For coatings that do not form stable membranes overextended periods of time, the fiber can be dipped into a solution of thecoating material. The device formats described above facilitate thecoating of fibers with the membrane forming liquids or solids by simpledipping of the cover plates carrying the fibers into the membraneforming solutions, as these cover plates are easily detachable from thewell plates. After coating, the cover plates can be put back on to thewell blocks, after washing off excess coating material sticking to thefiber. If the membrane forming material is a solid, a solution of themembrane forming solid in an appropriate solvent can be introduced intoa container such as a vial or well plate either manually or through anautomatic dispenser. A membrane forming liquid material can be used assuch for coating the fiber. A supported liquid membrane can be polar orhydrophobic in nature, depending upon the chemical nature of the analytebeing extracted. Polyethers, polyesters, polyurethanes, polyamides,polyvinylalcohol, polyalkylene glycols and polyacrylonitrile derivativescan be used to form polar coatings, to mention a few examples.Hydrophobic coatings include, but are not restricted to, hexadecane,polyalkylenes, polyalkenes with phenylenyl moieties. Each one of thefibers in the 48 or 96 or 384 well plate device format can be coatedwith a different membrane material, if needed.

Acceptor Phase Selectivity:

When extraction of basic pharmaceuticals from aqueous solutions or humanfluids is carried out, the sample solutions are rendered basic to keepthe drugs in the free state. A variety of acidic acceptor solutions areavailable for extraction of these basic drugs, such as the mineral acids(hydrochloric, nitric, sulfuric), organic acids (formic, acetic,propionic acids) or acidic buffers (such as phosphate or acetate orcitrate buffers whose pH has been adjusted to be in the range 2.0 to5.0). However, one is not restricted to these alone, and can use a widerselection of acidic materials. Strong acids may not be suitable for usewith silica-based solid phase extraction sorbents, since the bondedphases can be cleaved off under such conditions. The current membranebased devices have this clear advantage over the silica-based SPE bondedphases. Tables 1-A through 1-C show extraction recovery data for sevenbasic drugs making use of 16 acidic acceptor solutions. Tables 1-A and1-B show extraction recovery values and enrichment values, respectively.Table 1-C shows extraction recovery values averaged over the sevendrugs.

TABLE 1-A Extraction recovery with different acceptor phases Extractionrecovery (average of 3 replicates) Acceptor phase Measured pH #1 #2 #3#4 #5 #6 #7  10 mM HCl 2.1 51% 76% 80% 79% 86% 83% 43% 100 mM HCl 1.246% 75% 87% 86% 84% 88% 53%  10 mM H₂S0₄ 2.1 65% 88% 90% 76% 98% 87% 35%100 mM H₂S0₄ 1.3 61% 84% 88% 85% 94% 99% nd  10 mM HNO₃ 1.9 30% 78% 66%75% 88% 85% 49% 100 mM HNO₃ 1.1 nd nd 40% 85% 67% 61% 39%  10 mM H₃PO₄2.5 45% 60% 61% 74% 60% 61% 30% 100 mM H₃PO₄ 1.8 36% 52% 56% 50% 48% 56%29%  10 mM HCOOH 3.1 45% 66% 72% 45% 71% 70%  8% 100 mM HCOOH 2.3  3% 8% 66% 58% 57% 62% 31%  10 mM CH₃COOH 3.3 41% 58% 60% 20% 54% 54%  5%100 mM CH₃COOH 2.7 30% 48% 56% 41% 53% 53% 12%  10 mM phosphate 3.3 77%85% 87% 52% 84% 84% 12% 100 mM phosphate 3.0 80% 83% 82% 58% 73% 80% 32% 10 mM acetate 4.8 56% 80% 57%  2% 26% 24% nd 100 mM acetate 4.8 is is66% is 26% 25% nd #1 = amphetamine, #2 = methamphetamine, #3 =pethidine, #4 = chlorcyclizine, #5 = methadone, #6 = haloperidol, and #7= buprenorphine; nd = not detectable, is = insufficient separation foraccurate quantitation

TABLE 1-B Enrichment with different acceptor phases Enrichment (averageof 3 replicates) Acceptor phase Measured pH #1 #2 #3 #4 #5 #6 #7  10 mMHCl 2.1 82 122 128 126 138 133 69 100 mM HCl 1.2 74 120 139 138 134 14184  10 mM H₂S0₄ 2.1 104 141 144 122 157 139 56 100 mM H₂S0₄ 1.3 98 134141 136 150 158 nd  10 mM HNO₃ 1.9 48 125 106 120 141 136 78 100 mM HNO₃1.1 nd nd 64 136 107 98 62  10 mM H₃PO₄ 2.5 72 96 98 118 96 98 48 100 mMH₃PO₄ 1.8 58 83 90 80 77 90 46  10 mM HCOOH 3.1 72 106 115 72 114 112 13100 mM HCOOH 2.3 5 13 106 93 91 99 50  10 mM CH₃COOH 3.3 66 93 96 32 8686 8 100 mM CH₃COOH 2.7 48 77 90 66 84 84 19  10 mM phosphate 3.3 123136 139 83 134 134 19 100 mM phosphate 3.0 128 133 131 93 117 128 51  10mM acetate 4.8 90 128 91 3 42 38 nd 100 mM acetate 4.8 is is 106 is 4240 nd #1 = amphetamine, #2 = methamphetamine, #3 = pethidine, #4 =chlorcyclizine, #5 = methadone, #6 = haloperidol, and #7 =buprenorphine; nd = not detectable, is = insufficient separation foraccurate quantitation

TABLE 1-C Average recovery for 7 drugs with different acceptor phasesAverage extraction Average extraction Acceptor phase recovery Acceptorphase recovery  10 mM HCl 71%  10 mM HCOOH 54% 100 mM HCl 74% 100 mMHCOOH 41%  10 mM H₂S0₄ 77%  10 mM CH₃COOH 42% 100 mM H₂S0₄ 73% 100 mMCH₃COOH 42%  10 mM HNO₃ 67%  10 mM phosphate 69% 100 mM HNO₃ 42% 100 mMphosphate 70%  10 mM H₃PO₄ 56%  10 mM acetate 35% 100 mM H₃PO₄ 47% 100mM acetate 39%

The data in Tables 1-A through 1-C was generated by performing LPMEusing three different hollow fibers. The extractions were performed fromwater samples containing each component at the 100 ng/mL level.

Significant differences could be detected between the acids studied asacceptors. In general, mineral acids and phosphate buffers of low pHfurnished the highest recoveries for the drugs. Lower recoveries wereobtained with acetic and formic acids and acetate buffers and thediscrepancies could be attributed to variation in acceptor phase pH,buffer capacity or the solubility of drugs with different counter ions.It is evident that a selective enrichment between basic drugs could beachieved by controlling the acceptor phase chemistry. Theelectropherograms of the seven drugs with different acidic acceptors areincluded in FIG. 9. The peaks labeled 1-7 in FIG. 9 correspond to thedrugs labeled 1-7 in Table 1-A.

Selectivity from Acceptor Phase pH:

It is possible to vary the pH of the acceptor phase, during theextraction of basic drugs, by changing the pH of the buffer. FIG. 10shows the electropherograms of eight basic drugs obtained by analyzingthe extracts obtained by using phosphate buffers ranging from pH 2.5 to7.5 as acceptor phases. Tables 2-A and 2-B show the selectivity obtainedwith basic drugs when acceptors of different pHs are used. The peakslabeled 1-8 in FIG. 10 correspond to the drugs labeled 1-8 in Tables2-A-B.

TABLE 2-A Extraction recovery with different acceptor phases Extractionrecovery (average of 3 replicates) pH #1 #2 #3 #4 #5 #6 #7 #8 2.5 67%94% 100%  77% 5% 105% 2% 11% 3.5 64% 91% 92% 20% 1%  83% 1%  1% 4.5 65%94% 82%  3% nd  49% nd nd 5.5 58% 84% 56% nd nd  22% nd nd 6.5 55% 76%21% nd nd  4% nd nd 7.5 40% 55%  4% nd nd nd nd nd #1 = amphetamine, #2= methamphetamine, #3 = pethidine, #4 = chlorcyclizine, #5 = noscapin,#6 = haloperidol, #7 = diazepam, and #8 = reserpin; nd = not detectable

TABLE 2-B Enrichment with different acceptor phases Enrichment (averageof 3 replicates) PH #1 #2 #3 #4 #5 #6 #7 #8 2.5 107 150 160 123 8 168 318 3.5 102 146 147  32 2 133 2  2 4.5 104 150 131  5 nd  78 nd nd 5.5 93134 90 nd nd  35 nd nd 6.5 88 122 34 nd nd  6 nd nd 7.5 64 88 6 nd nd ndnd nd #1 = amphetamine, #2 = methamphetamine, #3 = pethidine, #4 =chlorcyclizine, #5 = noscapin, #6 = haloperidol, #7 = diazepam, and #8 =reserpin; nd = not detectable

The extraction recovery and enrichment data demonstrates that at pHvalues below 3.0, all the basic drugs are extracted substantiallycompletely. However, as pH increases progressively, there is asignificant change in extractability of these basic drugs, especiallybeyond pH 6.0 and this is attributable to differences in the pK_(a)values of the drugs investigated. These experiments clearly show that amixture of basic drugs can be selectively extracted from aqueousmatrices by controlling the pH of the acceptor phase.

Selectivity Based on Hollow Fiber Chemistry:

The differences in the hydrophobicity and polarity of the materials fromwhich the hollow fibers are generated could be utilized for impartingselectivity to the fiber during the extraction process. We investigatedpolypropylene and polysulfone fibers for their capacities for extractinga mixture of seven basic drugs under identical conditions. The resultingdata is presented in Tables 3-A and 3-B. Table 3-A lists measuredextraction recovery values, while Table 3-B lists measured enrichmentvalues. Capillary electrophoresis data is shown in FIG. 11. The peakslabeled 1-7 in FIG. 11 correspond to the drugs labeled 1-7 in Tables3-A-B.

TABLE 3-A Extraction recovery with different hollow fibres Extractionrecovery (average of 3 replicates) Hollow fibre #1 #2 #3 #4 #5 #6 #7Polypropylene, 51% 76% 80% 79% 86% 83% 43% 600 μm ID Polypropylene, 65%77% 76% 66% 86% 82% 61% 280 μm ID Polysulfone, 14% 35% 78% 69% 87% 54%58% 500 μm ID #1 = amphetamine, #2 = methamphetamine, #3 = pethidine, #4= chlorcyclizine, #5 = methadone, #6 = haloperidol, and #7 =buprenorphine

TABLE 3-B Enrichment with different hollow fibres Enrichment(average of3 replicates) Hollow fibre #1 #2 #3 #4 #5 #6 #7 Polypropylene,  82 122128 126 138 133 69 600 μm ID Polypropylene, 104 123 122 106 138 131 98280 μm ID Polysulfone, 500 μm ID  22  46 125 110 139  86 93 #1 =amphetamine, #2 = methamphetamine, #3 = pethidine, #4 = chlorcyclizine,#5 = methadone, #6 = haloperidol, and #7 = buprenorphine

A significant selectivity difference could be noticed in the case ofamphetamine and methamphetamine, with polysulfone exhibiting much lowerrecoveries. Further, a similar effect was also evidenced withhaloperidol, although to a much smaller extent.

Selectivity Based on Membrane Chemistry:

Little information was previously available on the differences in thebehavior of membranes in the separation process. We have used themultiple sampling device to demonstrate the selectivity of fourdifferent membrane liquids, i.e. hexyl ether, 2-octyl-1-dodecanol,1-octanol and 4-nitrophenyl octyl ether. A fifth material, N-octyl2-pyrrolidone, did not show good extraction capability for the testedapplication. A mixture of acidic and basic drugs was used in this study,together with 0.1 M hydrochloric acid or 0.1 M sodium hydroxide as theacceptor phase. The chromatograms presented in FIGS. 12-A-B along withthe data in Table 4 indicate that the four membranes have differentselectivity to the basic probes. FIG. 12-A shows a chromatographillustrating the enrichment of naproxen using a nitrophenyl octylethermembrane and a 0.1 M sodium hydroxide acceptor. FIG. 12-B shows achromatograph illustrating the enrichment of doxepin and quinidine witha nitrophenyl octylether membrane and a 0.1 M hydrochloric acidacceptor. The basic drugs are preferentially extracted into thehydrochloric acid acceptor from a basified sample solution, while theacidic drugs are selectively extracted into the sodium hydroxideacceptor from an acidified sample solution.

TABLE 4 Enrichment of Quinine and Doxepin on Different Liquid MembranesSupported Liquid Membrane Enrichment of Material Enrichment of QuindineDoxepin Hexyl Ether 100 202 4-nitrophenyl octyl ether 52 227 1-octanol100 58 2-octyl-1-dodecanol 20 147Operation of the Devices and Sample to Acceptor Volume Ratios:

The devices described above operate in a static mode, as opposed to amode in which the acceptor solution circulates through the membranefibers. In a static mode, the sample and/or acceptor solution may bevibrated inside their container(s), but do not flow through the fiber.The extractions are typically completed within 15 to 30 minutes,depending upon the nature of the sample. With whole blood or plasmasamples, about 30 minutes may be used to complete the extraction step.With simpler aqueous sample solutions, extraction times as low as 5minutes can be sufficient to attain equilibrium between the samplesolution and the acceptor phase.

The donor sample preferably has a volume higher than 200 μL and lowerthan 25 ml. Sample volumes on the order of 500 μL can be readilyemployed. The acceptor solution preferably has a volume higher than 10μL and lower than 500 μL. Acceptor solution volumes volumes lower than100 μL can be readily employed, and acceptor volumes between 20 and 50μL are commonly utilized. If the fiber dimensions are as small as 4 to 5cm, 10 μL of acceptor can be sufficient. Longer fibers can hold largeramounts of acceptor solution. The current device facilitates the use offibers of any desired dimension. The length of the employed fiber ispreferably between 1 cm and 20 cm, and is commonly longer than 2 cm. Incommon implementations, the inner diameter of the fiber is between 0.3mm and 1.5 mm, and preferably between 0.6 mm and 1.2 mm. The hollowfiber has an average pore size in a range between 0.02 μm and 2 μm. Apresent implementation employs fibers with lengths of 7 to 8 cm, 500micron inner diameter, 0.2 micron pore size, and an acceptor phasevolume of 25 μL.

The ratio between the sample solution to acceptor solution volume canvary typically from 20 to 200, while equilibration times can still be inthe 15 to 30 minute range. Adjustment of the acceptor solution volumecan be used to control the sample enrichment. This ratio can becontrolled by employing fibers of appropriate length or thicker fiberscan be made use of if larger acceptor volumes are needed to be used.

If a collection needle comes into close proximity with a hollow fiber,it is preferred that the needle diameter be less than half the size ofthe internal diameter of the hollow fiber, such that the collectionneedle does not damage or puncture the fiber. Increasing the diameter ofa hollow fiber may require increasing its wall thickness, in order topreserve the mechanical stability of the fiber. At the same time,increasing the fiber wall thickness can lead to unacceptably long timeperiods required to achieve desired levels of enrichment. For typicalfiber compositions, it was observed that fiber diameters larger thanabout 1.2 mm may require fiber walls thicker than about 200 μm formechanical stability. At the same time, increasing the fiber wallthickness to over about 200 μm was observed to lead to a marked increasein the time required to achieve useful levels of enrichment.

EXAMPLES

I. Separation and Enrichment of Pharmaceuticals from Human Fluids:

(1) Methamphetamine in human plasma and urine with the vial formatdevice: a polypropylene fiber (8.0 cm long, 600 μm inner diameter, 0.2μm pore size), obtained from Akzo Nobel and sold under the name AccurelPP Q3/2, was connected to a syringe needle (0.81 mm inner diameter)carrying a needle guide head on one end and to a syringe needle of thesame dimension without the guide head on the other end. The fiber wasdipped into pure 1-octanol contained in a 20 mL glass vial for about 5seconds. The fiber was then withdrawn and dipped into deionized watercontained in a separate 20 mL glass vial and sonicated for 15 seconds.25 μL of 0.1M hydrochloric acid was injected into the lumen side of theabove fiber with a syringe. In the meanwhile, a sample solution wasprepared by treating 2.5 mL of the urine or plasma sample containingmethamphetamine with 125 μL of 2.0 M sodium hydroxide. The fibercontaining the acceptor acid solution was then dipped into this samplesolution and the vial was shaken on a Vibramax 100 shaker for 45minutes. The acceptor solution was then collected into a clean vial bypushing air under pressure from the needle guide head side of the fiberwith a syringe and placing a clean microvial at the other end of thefiber. The collected enriched and purified sample in the acceptorsolution was then subjected to capillary zone electrophoesis (CZE).Conditions for CZE were 50 mM phosphate (pH 2.75) running buffer, 15 kVseparation voltage, 30 cm effective length/75 μm inner diametercapillary tube and UV detection at 200 nm. An extraction efficiency of75%, together with an enrichment of 75 fold was obtained and thedetection limit was 5 ng/mL. The RSD from six experiments was found tobe 5.2%. The resulting electropherograms are shown in FIGS. 13-A-B. FIG.13-A shows an electropherogram for LPME/CZE of 100 ng/ml of methamphineextracted from human urine, while FIG. 13-B shows an electropherogramfor LPME/CZE of 100 ng/ml methamphetamine extracted from human plasma.

(2) Naproxen from human urine with the vial format device: apolypropylene fiber, attached to a pair of syringe needles as outlinedin example 1, was dipped in hexyl ether for 5 seconds and then sonicatedin deionized water for 15 seconds to remove excess hexyl ether adheringto the fiber. Then, 25 μL of a 3:1 mixture of 0.01 M sodiumhydroxide/methanol was injected into the lumen side of the fiber. Thefiber was dipped into a sample solution consisting of 2.5 μL of urinecontaining the non-steroidal anti-inflammatory drug naproxen to which250 μL of 1 M hydrochloric acid has been added. After 45 minutes, theacceptor solution was recovered and subjected to capillary zoneelectrophoresis with 30 mM acetate (pH 4.75) as running buffer, using aseparation voltage of 20 kV, a capillary of 30 cm effective length/75 μminner diameter and a detection wavelength of 226 nm. An enrichment of 82fold, along with a recovery of 82% was observed. The RSD from sixexperiments was 4.6% and the detection limit was 2 ng/mL. The resultingelectropherogram is shown in FIG. 14.

(3) Citalopram and its N-desmethyl metabolite from human plasma with thevial format device: a polypropylene fiber, connected to a pair ofsyringe needles as outlined in example 1, was coated with hexyl etherfor 5 seconds and then sonicated in deionized water for 15 seconds toremove excess hexyl ether adhering to the fiber. A 20 mM phosphatebuffer solution (pH 2.75, 25 μL) was used as the acceptor solution onthe lumen side of the fiber. The fiber was dipped into a mixture of 1 mLof plasma containing 2.73 mL of water and 250 μL of 2 M sodiumhydroxide. The plasma sample was obtained from a patient treated with 40mg of citalopram daily. The recovered acceptor phase after 45 minutes ofextraction was analyzed by CZE with 75 mM TRIS-acetic acid (pH 4.6)containing 3% weight/volume of Tween 20 and 75 mg/L of FC-135 as runningbuffer on a 40 cm capillary column using a detector wavelength of 200nm. A preconcentration of 30 fold, together with an extraction recoveryof 75% could be observed. The RSD from six experiments was 3.6%, with adetection limit of 5 ng/mL. FIG. 15 shows the resultingelectropherogram.

Citalopram and methamphetamine in human whole blood employing the vialformat device: a polypropylene fiber (280 μm inner diameter, 27 cmlength, 0.2 μm pore size and 50 μm wall thickness) was coated with hexylether as mentioned in the above examples 2 and 3. The fiber was dippedinto 2.5 mL of whole blood containing 1.125 mL water and 125 μL of 2 Msodium hydroxide. Using 17 μL of 0.1 M hydrochloric acid as acceptor,the extraction of the drugs from whole blood was done for 30 minutes.The recovered acceptor solution was analyzed by CZE with 50 mM acetate(pH 4.6) as running buffer, a separation voltage of 15 kV and a detectorwavelength of 200 nm on a capillary column of 30 cm effective length. Aone hundred fold enrichment of the two drugs was observed. The resultingelectropherogram is included in FIG. 16. The upper graph in FIG. 16corresponds to whole blood containing drugs, while the lower graphcorresponds to drug-free whole blood.

Tramadol from human plasma through vial format device: a polypropylenefiber (with dimensions same as in example 4), coated with hexyl ether,was dipped into 0.5 mL of plasma containing 3.25 mL water and 250 μL of2 M sodium hydroxide for 45 minutes. An acceptor solution of 0.1 Mhydrochloric acid (17 μL) was used. Analysis of the enriched acceptorphase was done by CZE with a running buffer consisting of 50 mMphosphate buffer pH 2.5+5 mM carboxymethyl-β-cyclodextrin at 200 nm and20 kV on a capillary of 50 cm effective length. An enrichment of 30 foldwith extraction efficiency of 100% was observed. FIG. 17 shows theresulting electropherogram.

Mianserine from human plasma with a vial format device: a polypropylenefiber of the same dimensions as in example 4 was used in this experimentcarried out in the same fashion as described under example 5, exceptthat a running buffer of 75 mM phosphate buffer (pH 3.0)+triethylamineand 2 mM hydroxypropyl-β-cyclodextrin was used. The enrichment wasobserved to be 15 fold and an extraction efficiency of 50% wasregistered. The resulting electropherogram is shown in FIG. 18.

Methamphetamine, pethidine, promethazine, methadone and haloperidol fromhuman plasma and whole blood with a vial format device: a polypropylenefiber (8 cm, 600 μm inner diameter, 0.2 μm pore size) suspended in avial format device was treated with a sample solution comprising of 250μL of plasma/whole blood, 250 μL of 2.0 M sodium hydroxide and 500 μL ofwater for 30 minutes. The fiber is coated with hexyl ether membrane andcarried 25 μL of 0.01 M hydrochloric acid as acceptor phase. Therecovered acceptor solution was subjected to capillary zoneelectrophoresis with 25 mM phosphate (pH 2.75) as running buffer, 30 kVseparation voltage, 200 nm detection wavelength and a 50 cm capillary.Extraction efficiencies of about 55-80% were obtained depending upon thenature/chemistry of the drug, together with enrichment of 6 to 8 fold.The resulting electropherogram is shown in FIG. 19, while Table 5 listsextraction efficiency and enrichment values for the five compounds inplasma and whole blood samples.

TABLE 5 Enrichment of Quinine and Doxepin on Different Liquid MembranesExtraction efficiency/enrichment Compound Plasma Whole bloodMethamphetamine 81%/8.1 78%/7.8 Pethidine 74%/7.4 72%/7.2 Prometazine55%/5.5 43%/4.3 Methadone 64%/6.4 54%/5.4 Haloperidol 67%/6.7 55%/5.5

Amphetamine from human urine with vial format device: a polypropylenefiber (dimensions, membrane coating and acceptor chemistry as in example7) was suspended in 2.0 mL of urine containing 250 μL of 2.0 M sodiumhydroxide and 2.0 mL of water for 45 minutes with vibration. Theresulting acceptor solution was analyzed by capillary electrophoresisunder the same conditions described in example 7. Extraction efficiencyof 97% and enrichment of 77 was observed. The resulting electropherogramis shown in FIG. 20.

Chlorcyclizine from human plasma with vial format device: apolypropylele fiber (dimensions, membrane coating and acceptor phase asin example 7) was suspended in 2.0 mL of plasma containing 250 μL of 2.0M sodium hydroxide and 2.0 mL of water for 45 minutes with vibration.The enrichment was 52 and recovery 65%. The resulting electropherogramis shown in FIG. 21.

II. Selectivity Through Acceptor Phase Variation:

A sample solution was prepared by mixing solutions of seven basic drugscontaining the drugs at 100 ng/mL concentration (100 μL each) anddiluting to 4 mL with water. These drugs consist of amphetamine,methamphetamine, pethidine, chlorcyclizine, methadone, haloperidol andbuprenorphine. The pH of the solution was adjusted to be on the basicside by adding 250 μL of 2.0 M sodium hydroxide. A polypropylene hollowfiber was coated with dihexylether to form a supported liquid membranein the pores of the fiber. The dimensions of the fiber are the same asindicated in Example 1 under Section I. This fiber was dipped into theabove seven component drug mix taken in the vial format device and theextraction was allowed to proceed for 60 minutes with shaking by aVibramax 100 vibrator. The acceptor fluid inside the fiber was 25 μL ofthe appropriate acid solution listed in Table 1-A. At the conclusion ofthe extraction period, the acceptor solution was recovered in the mannerdescribed under Example 1 (Section I) and subjected to capillaryelectrophoresis with 25 mM phosphate (pH 2.75) as running buffer, 30 kVseparation voltage, 200 nm detector wavelength and a 60 cm capillarycolumn. The results are presented in FIG. 9 and Tables 1-A through 1-C.Recoveries of around 70% could be obtained when hydrochloric, sulphuricand nitric acids and phosphate buffers of pH 1.8 and 2.5 were used asacceptors. On the other hand, with acetic and formic acids as acceptors,the recoveries were in the 40-50% range. Furthermore, enrichment factorsof over 120 were recorded with the strong acids and strongly acidicphosphate buffers. The selectivity of different acceptor acids isdemonstrated by the fact that for methadone, the enrichment was 138 withhydrochloric acid, while it drops down to 42 with acetate buffer of pH4.8. On the other hand, for pethidine, the acetate buffer shows anenrichment of 106, while nitric acid shows a figure of 64.

III. Selectivity Through Acceptor Phase pH Variation:

An eight component drug mixture consisting of amphetamine,methamphetamine, pethidine, chlorcyclizine, noscapin, haloperidol,diazepam and reserpine was used. A mixture of 100 μL of each drug(originally at 100 ng/mL concentration) was diluted to 4.0 mL with waterand pH of the resulting solution adjusted to the basic side with 2.0 Msodium hydroxide (250 μL). The acceptor solutions were 10 mM phosphatebuffers whose pH was adjusted to be 2.5, 3.5, 4.5, 5.5, 6.5 and 7.5,respectively. The fiber dimensions were as described above forexample 1. An extraction time of 60 minutes was used. The results ofcapillary electrophoresis (see FIG. 10 and Tables 2-A-B) show thatselective extraction and enrichment of all the eight drugs could be madeat lower pH values, while the values drop off starting from pH 5.0.

IV. Selectivity Based on Fiber Chemistry:

The seven-component drug mix described above in Section II was utilized.The extraction experiments were performed on polypropylene fibers of 600μm inner diameter, 8 cm length and 0.2 μm pore size and on polysulfonefibers of 500 μm inner diameter, 8 cm length and 0.2 μm pore size. Bothtypes of fibers were coated under identical conditions with hexyl ether.Details of sample solution generation are the same as in Section IIabove. The acceptor solution was 0.01 M hydrochloric acid. FIG. 11 andTables 3-A-B show the data from these experiments. The selectivitybetween the fibers is evident from methamphetamine which is enriched tobe extent of 82% on polypropylene, while the same drug is recovered tothe extent of only 22% on polysulfone. On the other hand, buprenorphinewas enriched to the tune of 93% on polysulfone, while the figure forpolypropylene is 69%.

V. Selectivity Based on Membrane Chemistry using the Well Format Device:

Four different membrane forming small molecular weight organic liquidswere investigated for selectivity differences, viz. hexyl ether,4-nitrophenyl octyl ether, 1-octanol and 2-octyl-1-dodecanol. The firstbelongs to an aliphatic ether type, while the second is an aryl alkylether containing the polar nitro functionality. The last two are fromthe aliphatic alcohols variety, but 1-octanol is a straight chainmolecule as opposed to the dodecanol which is a branched chain (andlonger) molecule. A cover plate of a 96 well block, carryingpolypropylene fiber of 8 cm length, 600 μm inner diameter and 0.2 μmpore size, was dipped into each of these pure liquids for 5 seconds.These liquids were contained in different wells of a 96 well plate. Thefibers in the cover plate were then washed by sonication in water for 15seconds to remove excess material sticking to the fibers. A fivecomponent mixture of acidic and basic drugs was prepared fromacetaminophen, naproxen, bamethane, quinidine and doxepin.Concentrations of the stock drug solutions were 1.0 mg/mL in each case.However, the drugs which have strong absorption in the UV were taken insmaller amounts—20 μL each of acetaminophen, naproxen and doxepin in themixture, while the other two drugs were taken in larger amounts (100 μLeach). This is to maintain roughly the same level of analytical signalwith each of these drugs. The mixture was diluted to 20 mL with water sothat the concentrations of the three dilute drugs is in the range of 1μg/mL, while those of the more concentrated drugs is in the range of 5μg/mL. 500 μL of this diluted mixture of drugs is further diluted eightfold (to 4 mL) with water containing 250 μL of 2.0 M sodium hydroxideand used for extraction. Thus, the concentration of acetaminophen,naproxen and doxepin in the sample solution are about 60 ng and those ofquinidine and bamethan are about 300 ng. The acceptor solution consistedof 25 μL of 0.1 M hydrochloric acid in each case. Extraction time withvibration was 30 min for each membrane liquid. The enriched acceptorsolution was diluted three fold in each case and 25 μL of the resultingdiluted solution used for analysis by high performance liquidchromatography on a Omnisphere C18 column using acetonitrile/pH 7.0dipotassium hydrogen phosphate as mobile phase. A gradient from 5%acetonitrile to 40% was used to elute the strongly retained componentsin a reasonable time frame. The results included in Table 4 and FIG. 12demonstrate over 100 to 200 fold enrichments of the basic drugsquinidine and doxepin. In addition, each of the membrane materialsexhibits a different selectivity between quinidine and doxepin. Thus,1-octanol is selective towards quinidine (2:1 enrichment ratio forquinidine:doxepin), while 2-octyl 1-dodecanol shows a 7:1 enrichment infavor of doxepin. This demonstrates that even within the alcohol groupof membranes, depending upon the alkyl chain length one can manipulateselectivity. For nitrophenyl octyl ether, the enrichment ratiodoxepin:quinidine works out to 5:1, while for hexyl ether it is 2:1,which again demonstrates the difference in selectivity between aliphaticand aryl-alkyl ethers.

VI. Smaller Sample Volume: Acceptor Volume Ratios:

The sample solution consisted of promethazine, methadone and haloperidol(100 ng each) in 750 μL of water and 250 μL of sodium hydroxide (2.0 M).Hexyl ether is the membrane forming liquid on the polypropylene fiberand the acceptor solution was 0.01 M hydrochloric acid (25 μL). Thus,the sample:acceptor volume ratio is 40:1. The extractions were performedfor 2, 5, 10, 15 and 30 minutes, respectively. In a second set ofexperiments, an acceptor volume of 50 μL was used, so that thesample:acceptor volume ratio becomes 20:1. FIGS. 22-A-C show extractiontime profiles for prometazine, methadone, and haloperidol, respectively,for the first set of experiments. FIGS. 22-D-F show extraction timeprofiles for prometazine, methadone, and haloperidol, respectively, forthe second set of experiments. In both sets of experiments, it was foundthat equilibrium could be reached within 5 min, as illustrated in FIGS.22-A-F. This example demonstrates that devices and processes accordingto the present invention can work efficiently with either larger orsmaller sample:acceptor volume ratios.

It will be clear to one skilled in the art that the above embodimentsmay be altered in many ways without departing from the scope of theinvention. Although 96 well block formats are presented in the presentinvention, many other multi-well formats can be applied for the sameLPME purpose, such as 48, 24 or 384 well formats etc. Although only onesingle hollow fiber in each well or vial is pictured in the presentformats, multiple hollow fibers can be connected to each of the wells orvial caps. It is understood that any recited steps need not be performedin the exact order listed in a given claim. Accordingly, the scope ofthe invention should be determined following claims by the and theirlegal equivalents.

1. A sample purification and enrichment method comprising: inserting adonor sample in a well of a multi-well plate, the donor samplecomprising an analyte of interest; inserting a tubular hollow porousfiber into the well, the hollow fiber comprising a liquid extractionmembrane, the hollow fiber enclosing an internal cavity separated fromthe donor sample by the liquid extraction membrane; placing a staticacceptor liquid in the internal cavity; simultaneously enriching andcleaning up the analyte of interest by extracting the analyte ofinterest from the donor sample into the acceptor liquid in the internalcavity through the liquid extraction membrane; and transferring theanalyte of interest and the acceptor liquid from the internal cavity toan analysis device.
 2. The method of claim 1, wherein the multi-wellplate is a monolithic plate having fixed wells.
 3. The method of claim1, wherein the multi-well plate comprises a base plate having aplurality of apertures, and a plurality of removable vials each insertedthrough one of the plurality of apertures, each vial defining a well ofthe multi-well plate.
 4. The method of claim 1, wherein the multi-wellplate comprises a bottom block defining the well, and a topfiber-supporting plate mounted on the bottom block, the top platecomprising the fiber, the top plate having a through hole connected tothe internal cavity and aligned to the well.
 5. The method of claim 4,wherein the top plate further comprises a protective insert extendinglaterally around the fiber and having an open lower end, formechanically protecting the fiber.
 6. The method of claim 5, wherein theprotective insert is tapered such that the open lower end has a smallersize than an upper end of the insert.
 7. The method of claim 4, furthercomprising inserting a guiding pin of the top plate into a correspondingguiding aperture formed in the bottom block around the well, foraligning the fiber in the well, wherein each well of the multi-wellplate has at least one individually-corresponding guiding aperture. 8.The method of claim 1, wherein the hollow fiber is a rod-shaped fiber.9. The method of claim 1, wherein the hollow fiber is a U-shaped fiber.10. The method of claim 9, wherein the internal cavity is connected toan exterior of the multi-well plate through a single access openingformed in the multi-well plate.
 11. The method of claim 9, wherein theinternal cavity is connected to an exterior of the multi-well platethrough at least two access openings formed in the multi-well plate. 12.The method of claim 1, wherein the hollow fiber comprises twointerconnected, parallel longitudinal rods.
 13. The method of claim 1,further comprising, after extracting the analyte of interest into theinternal cavity, pushing the acceptor liquid and the analyte of interestinto an open container corresponding to the well, the container havingan inlet connected to the hollow fiber, and an upper outlet opening forallowing the transferring of the analyte of interest and the acceptorliquid from the container to the analysis device.
 14. The method ofclaim 1, further comprising pre-depositing the liquid extractionmembrane in the hollow fiber before placing the acceptor liquid in theinternal cavity.
 15. The method of claim 1, wherein the donor sample hasa volume higher than 200 μl and lower than 25 ml.
 16. The method ofclaim 15, wherein the acceptor liquid has a volume higher than 10 μl andlower than 500 μl.
 17. The method of claim 15, wherein the acceptorliquid has a volume lower than 100 μl.
 18. The method of claim 1,wherein a volume ratio of the donor sample to the acceptor liquid ishigher than 20 and lower than
 200. 19. The method of claim 1, whereinthe hollow fiber has an inner diameter equal or smaller than 1.2 mm andequal or larger than 0.6 mm.
 20. The method of claim 19, wherein thehollow fiber is longer than 1 cm and shorter than 20 cm.
 21. The methodof claim 19, wherein the hollow fiber has an average pore size equal orhigher than 0.02 μm and equal or lower than 2 μm.
 22. The method ofclaim 1, wherein the hollow fiber is formed substantially of a materialselected from a polymer, a cellulose derivative, a glass fiber, and aceramic.
 23. The method of claim 22 wherein the hollow fiber comprises amaterial selected from a polyolefin, a polysulfone,polytetrafluoroethylene, a polycarbonate, a polyetherketone,polystyrene, cellulose, cellulose acetate, polysiloxane, polyacrylate, apolyamide, and polyacrylonitrile.
 24. The method of claim 22, whereinthe sample is an organic sample, and the liquid extraction membrane isan aqueous membrane immiscible with the organic sample.
 25. The methodof claim 22, wherein the sample is an aqueous sample, and the liquidextraction membrane is an organic membrane immiscible with water. 26.The method of claim 25, wherein the liquid extraction membrane comprisesa material selected from an aliphatic hydrocarbon,. an aromatichydrocarbon, an ether, an ester, a nitrile, an aldehyde, a ketone, andan alcohol.
 27. The method of claim 1, wherein different wells of themulti-well plate hold hollow fibers having different chemistries. 28.The method of claim 1, farther comprising analyzing the analyte ofinterest after transferring the analyte of interest to the analysisdevice.
 29. The method of claim 28, wherein analyzing the analyte ofinterest comprises performing an analysis selected from a massspectrometry analysis and a chromatography analysis on the analyte ofinterest.
 30. The method of claim 1, wherein different wells of themulti-well plate hold liquid extraction membranes having differentchemistries.
 31. The method of claim 1, wherein different wells of themulti-well plate hold acceptor liquids having different pH values. 32.The method of claim 1, wherein different wells of the multi-well platehold acceptor liquids having different chemistries.
 33. A samplepurification and enrichment method comprising: simultaneously enrichingand cleaning up an analyte of interest by extracting the analyte ofinterest from a donor sample into a static acceptor liquid through aliquid extraction membrane formed in a wall of a porous hollow fibersituated in a well of a multi-well plate, the hollow fiber enclosing theacceptor liquid; and transferring the analyle of interest from thehollow fiber to an analysis device.
 34. A sample purification andenrichment method comprising: simultaneously enriching and cleaning upan analyte of interest by extracting the analyte of interest from adonor sample into a static acceptor liquid through a liquid extractionmembrane formed in a porous extraction disk situated in a well of amulti-well plate; and transferring the analyte of interest from the wellto an analysis device.
 35. A hollow-fiber membrane sample preparationmulti-well plate for enriching and cleaning up samples, comprising: aplurality of wells for holding a corresponding plurality of donorsamples, each donor sample comprising an analyte of interest; and aplurality of porous hollow fibers situated in the correspondingplurality of wells, each hollow fiber being situated in one of thewells, each hollow fiber including a liquid extraction membraneenclosing an internal cavity of the hollow fiber, for holding a staticacceptor liquid within each hollow fiber to receive the analyte ofinterest through the liquid extraction membrane into the acceptorliquid.
 36. The plate of claim 35, wherein the multi-well plate is amonolithic plate having fixed wells.
 37. The plate of claim 35, whereinthe multi-well plate comprises a base plate having a plurality ofapertures, and a plurality of removable vials each inserted through oneof the plurality of apertures, each vial defining a well of theplurality of wells.
 38. The plate of claim 35, wherein the multi-wellplate comprises a bottom block defining the plurality or wells, and atop fiber-supporting plate mounted on the bottom block, the top platecomprising the plurality of fibers, the top plate having an accessthrough hole connected to the internal cavity and aligned to the well.39. The plate of claim 38, wherein the top plate further comprises aprotective insert extending laterally around each fiber and having anopen lower end, for mechanically protecting said each fiber.
 40. Theplate of claim 38, wherein the protective insert is tapered such thatthe open lower end has a smaller size than an upper end of the insert.41. The plate of claim 38, wherein the top plate further comprises aplurality of guiding pins, each fiber corresponding individually to atleast one of the guiding pins, and wherein the bottom block comprises aplurality of guiding apertures defined between the plurality of wells,each guiding aperture being sized to receive a corresponding guiding pinfor aligning the plurality of fibers in the plurality of wells.
 42. Theplate of claim 35, wherein each hollow fiber is rod-shaped.
 43. Theplate of claim 35, wherein each hollow fiber is U-shaped.
 44. The plateof claim 43, wherein the internal cavity is connected to an exterior ofthe multi-well plate through a single access opening formed in themulti-well plate.
 45. The plate of claim 43, wherein the internal cavityis connected to an exterior of the multi-well plate through at least twoaccess openings formed in the multi-well plate.
 46. The plate of claim35, wherein each hollow fiber comprises two interconnected, parallellongitudinal rods.
 47. The plate of claim 35, further comprising aplurality of open collection containers each disposed above one of thewells, each collection container having an inlet connected to theinternal cavity, and an upper outlet opening.
 48. A hollow-fibermembrane sample preparation plate for enriching and cleaning up samples,comprising: a planar top plate having a plurality of access aperturesdefined therethrough, a spacing of the access apertures being chosensuch that each access aperture can be aligned to a well of asample-holding well block; and a plurality of porous hollow fibershanging from the planar top plate such that each access apertureprovides access to an internal cavity defined within one of the fibers,each hollow fiber including a liquid extaction membrane enclosing theinternal cavity, for holding a static acceptor liquid within each hollowfiber to receive an analyte of interest from a sample held in the wellthrough the liquid extraction membrane into the acceptor liquid.
 49. Asample preparation kit comprising: a multi well plate comprising aplurality of wells for holding a corresponding plurality of donorsamples each comprising an analyte of interest; and a top platecomprising a plurality of porous hollow fibers spaced apart so as to beinserted in the corresponding plurality of wells, each hollow fiberincluding a liquid extraction membrane for transferring the analyte ofinterest from the donor solvent to an acceptor liquid through the liquidextraction membrane.
 50. A hollow-fiber membrane sample preparationmulti-well plate for enriching and cleaning up samples, comprising: wellmeans comprising a plurality of wells for holding a correspondingplurality of donor samples, each donor sample comprising an analyte ofinterest; and hollow fiber support means for holding a plurality ofporous hollow fibers in the well means, each hollow fiber being situatedin one of the wells, each hollow fiber including a liquid extractionmembrane enclosing art internal cavity of the hollow fiber, for holdinga static acceptor liquid within each hollow fiber to receive the analyteof interest through the liquid extraction membrane into the acceptorliquid.